A monoclonal antibody targeting mesothelin and its application

Abstract

The present invention discloses a monoclonal antibody against mesothelin and its application. The monoclonal antibody against human mesothelin provided by the present invention or its antigen binding portion contains V H The heavy chain variable region and the name V L The light chain variable region, the V H and V L are composed of a complementary region and a framework region; the V H and the V L The complementary regions of the determinants are composed of CDR1, CDR2 and CDR3; H The CDR1, CDR2 and CDR3 of SEQ ID No: 5 are shown at positions 31-35, 50-66 and 99-109; the V L The CDR1, CDR2, and CDR3 of the mesothelin are shown at positions 24-34, 50-56, and 89-97 of SEQ ID No: 7. Since mesothelin is a new anti-tumor target with great research value, the anti-mesothelin monoclonal antibody prepared by the present invention can be used as a new ideal carrier for targeted anti-tumor drugs, laying a solid foundation for the research and development of targeted anti-tumor drugs and having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a monoclonal antibody targeting mesothelin and its application. Background Art

[0002] During the exploration of monoclonal antibody therapy for solid tumors, mesothelin (MSLN) was first discovered to be specifically expressed in ovarian cancer. The human mesothelin gene is located on chromosome 16p13.3, is approximately 8 kb in length, and consists of 16 exons totaling 7733 bp. The mesothelin precursor protein is a 69 kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that can be proteolytically cleaved at arginine 295 (Arg 295) into two components: a 30 kDa megakaryocyte potentiating factor (MPF) and a 40 kDa cell-adhesive mesothelin membrane-bound fragment. This fragment can also be shed from the cell surface by the tumor necrosis factor-converting protease. Mesothelin is rarely expressed in normal tissues but is highly expressed in various tumors, including malignant mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer. The biological function of mesothelin is still unclear. Mesothelin gene knockout mice do not show any detectable phenotype, and both male and female mice can reproduce normal offspring, which also indicates that mesothelin is a non-essential protein for growth and development. Mesothelin plays an important role in tumor cell proliferation, adhesion and drug resistance. Therefore, it can be used as a relevant tumor marker for early diagnosis of cancer, evaluation of cancer treatment effects, detection of the distribution of primary tumors and metastatic lesions, and become a new therapeutic target for tumor immunotherapy (J Clin Oncol. 2016 Dec 1; 34(34): 4171–4179.).

[0003] In recent years, research on mesothelin-targeting anti-tumor drugs, such as antibodies, antibody-drug conjugates (ADCs), immunotoxins, and tumor vaccines, has steadily advanced, with ADC research in particular making rapid progress. DMOT4039A, an ADC currently in Phase II clinical trials, is a conjugated humanized anti-mesothelin monoclonal antibody, h7D9.v3, to the anti-tubulin drug MMAE. This drug has demonstrated promising anti-tumor effects and is clinically used in the treatment of breast and ovarian cancers, with an objective response rate of 38% and a clinical efficacy rate of 55%. The highest dose is also well tolerated by patients. Therefore, DMOT4039A exhibits excellent safety and anti-tumor activity, and holds great promise for future development.

[0004] Hybridoma fusion technology was pioneered by Kohler and Milstein in 1975, and they were awarded the Nobel Prize in Biology and Medicine in 1984. Because B cells cannot proliferate indefinitely and survive in vitro for no more than 20 days, they are unable to produce monoclonal antibodies on a large scale. Tumor cells, on the other hand, can proliferate and survive indefinitely. Therefore, B cells capable of producing specific antibodies are fused (or electrofused) with myeloma cells using the fusion agent PEG (polyethylene glycol). Multiple positive clone screenings are then performed to obtain monoclonal hybridoma cell lines that stably secrete antibodies against specific antigens. The antibodies produced are monoclonal antibodies targeting a specific antigenic determinant and are characterized by high specificity, high purity, good homogeneity, high affinity, high titer, and low cost. In recent years, monoclonal antibody-based targeted tumor therapy has been considered one of the most promising and highly anticipated tumor treatment strategies. According to statistics, as of 2018, the FDA has approved a total of 24 monoclonal antibodies for the treatment of solid tumors. These drugs target CD antigens (including CD19, CD20, CD30, CD33, CD38, and CD53), tumor cell surface molecules (HER2, EGFR, PD-L1, GD2, PMSA, and SLAMF7), inhibitory receptors PD-1 and CTLA-4 on the surface of immune cells, and inhibit tumor angiogenesis. However, the current target range for monoclonal antibody drug development is very limited. Therefore, it is of great significance to discover new tumor-specific antigens, especially those that are highly expressed in tumor tissues and play an important role, and to develop monoclonal antibodies targeting these antigens to expand the application of monoclonal antibodies in the field of targeted tumor therapy.

[0005] Lidamycin (LDM) is a highly effective antitumor antibiotic consisting of a cofactor protein (LDP) and a chromophore (AE). LDP can be prepared through genetic engineering. Its spatial structure forms a hydrophobic pocket that protects the active enediyne chromophore (AE). LDP and AE are bound by a non-covalent bond, which is specific and robust, and can be disassembled and reconstructed. LDM's unique molecular structure makes it suitable as a "warhead" drug. Summary of the Invention

[0006] The purpose of the present invention is to provide a monoclonal antibody targeting mesothelin and its application.

[0007] The present invention provides an anti-human mesothelin monoclonal antibody or an antigen-binding portion thereof, characterized in that: the monoclonal antibody or the antigen-binding portion thereof contains V H The heavy chain variable region and the name V L The light chain variable region, the V H and V L are composed of a complementary region and a framework region; the V H and the VL The complementary regions of the determinants are composed of CDR1, CDR2 and CDR3;

[0008] The V H The amino acid sequence of CDR1 is shown in positions 31-35 of SEQ ID No: 5;

[0009] The V H The amino acid sequence of CDR2 is shown in positions 50-66 of SEQ ID No: 5;

[0010] The V H The amino acid sequence of CDR3 is shown in SEQ ID No: 5, positions 99-109;

[0011] The V L The amino acid sequence of CDR1 is shown in positions 24-34 of SEQ ID No: 7;

[0012] The V L The amino acid sequence of CDR2 is shown in positions 50-56 of SEQ ID No: 7;

[0013] The V L The amino acid sequence of CDR3 is shown in positions 89-97 of SEQ ID No: 7.

[0014] The naming system of the CDRs is the Kabat system.

[0015] The monoclonal antibody against human mesothelin can be a humanized monoclonal antibody, which is obtained by humanizing a mouse monoclonal antibody.

[0016] Specifically, the heavy chain variable region of the monoclonal antibody against human mesothelin is shown as positions 1-120 of SEQ ID No: 5, and the light chain variable region thereof is shown as positions 1-104 of SEQ ID No: 7.

[0017] Specifically, the heavy chain of the monoclonal antibody against human mesothelin is shown in SEQ ID No: 5, and the light chain thereof is shown in SEQ ID No: 7.

[0018] The anti-human mesothelin monoclonal antibody may be a mouse monoclonal antibody.

[0019] In mouse monoclonal antibodies, the V H and the V L The framework regions are derived from mouse.

[0020] Specifically, the heavy chain variable region of the monoclonal antibody against human mesothelin is shown as positions 20-139 of SEQ ID No: 1, and the light chain variable region thereof is shown as positions 21-127 of SEQ ID No: 3.

[0021] Specifically, the heavy chain variable region of the monoclonal antibody against human mesothelin is shown in SEQ ID No: 1, and the light chain variable region thereof is shown in SEQ ID No: 3.

[0022] The present invention also protects biological materials that are (a), (b), (c), (d) or (e):

[0023] (a) a nucleic acid molecule encoding any of the above monoclonal antibodies or antigen-binding portions thereof;

[0024] (b) an expression cassette comprising the nucleic acid molecule of (a);

[0025] (c) a recombinant vector having the nucleic acid molecule described in (a);

[0026] (d) a recombinant microorganism having the nucleic acid molecule described in (a);

[0027] (e) A transgenic cell line having the nucleic acid molecule described in (a).

[0028] The nucleic acid molecule encoding the heavy chain variable region may specifically be as follows (f1) or (f2):

[0029] (f1) shown in positions 58-417 of SEQ ID No: 2;

[0030] (f2) shown in SEQ ID No: 2.

[0031] The nucleic acid molecule encoding the light chain variable region may specifically be as follows (f3) or (f4):

[0032] (f3) shown in positions 61-381 of SEQ ID No: 4;

[0033] (f4) SEQ ID No: 4.

[0034] The nucleic acid molecule encoding the heavy chain variable region can be specifically shown as positions 1-360 of SEQ ID No: 6.

[0035] The nucleic acid molecule encoding the light chain variable region can be specifically shown as positions 1-312 of SEQ ID No: 8.

[0036] The nucleic acid molecule encoding the heavy chain can be specifically shown as SEQ ID No: 6.

[0037] The nucleic acid molecule encoding the light chain can be specifically shown as SEQ ID No: 8.

[0038] The present invention also protects hybridoma cell QJW-520-3B7, which was deposited on June 25, 2019 at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the deposit registration number CGMCC No. 18168.

[0039] The present invention also protects the monoclonal antibody secreted by the hybridoma cell QJW-520-3B7.

[0040] The present invention also protects the use of any of the above monoclonal antibodies, or any of the above antigen-binding portions, or any of the above hybridoma cells, or any of the above biomaterials, which is as follows (I) or (II) or (III) or (IV):

[0041] (I) Use in the preparation of vectors for targeting tumors or tumor cells;

[0042] (II) Application in the preparation of products for detecting mesothelin;

[0043] (III) Use in the preparation of products for detecting tumor tissues or tumor cells;

[0044] (IV) Use in the preparation of products for inhibiting tumors or tumor cells.

[0045] The present invention also protects a product, the active ingredient of which includes any of the above-mentioned monoclonal antibodies or any of the above-mentioned antigen-binding portions;

[0046] The product is as follows (i) or (ii) or (iii):

[0047] (i) Vectors for targeting tumors or tumor cells;

[0048] (ii) Products for the detection of mesothelin;

[0049] (iii) Products for detecting tumor tissue or tumor cells.

[0050] The present invention also protects a conjugate of an antibody and a drug; the antibody is any of the above-mentioned monoclonal antibodies or any of the above-mentioned antigen-binding portions.

[0051] Specifically, the drug may be lidamycin.

[0052] The conjugate can be specifically assembled from an antibody heavy chain, an LDP-fused antibody light chain, and a lidamycin chromophore.

[0053] The antibody heavy chain has a heavy chain variable region and a heavy chain constant region.

[0054] The heavy chain variable region may be any of the heavy chain variable regions described above.

[0055] Specifically, the heavy chain variable region is shown in SEQ ID No: 9.

[0056] The light chain of the LDP fusion antibody contains LDP, a light chain variable region, and a light chain constant region.

[0057] LDP, the cofactor protein of lidamycin.

[0058] Specifically, the LDP is shown as positions 22-131 of SEQ ID No: 11.

[0059] The light chain variable region may be any of the light chain variable regions described above.

[0060] Specifically, the light chain of the LDP fusion antibody is as shown in positions 22-243 of SEQ ID No: 11 or as shown in SEQ ID No: 11.

[0061] Specifically, the chromophore of lidamycin is as shown in formula (I).

[0062]

[0063] The preparation method of the conjugate may specifically be:

[0064] (1) The small fragment between the NheI and XhoI enzyme recognition sequences of the plasmid pIZDHL was replaced with the DNA molecule shown in sequence 10 of the sequence listing, and the small fragment between the MluI and BsiWI enzyme recognition sequences was replaced with the DNA molecule shown in sequence 12 of the sequence listing, while keeping the other sequences unchanged, to obtain the recombinant plasmid pIZDHL-MSLN-IgG-LDP;

[0065] (2) The recombinant plasmid pIZDHL-MSLN-IgG-LDP was digested with pvuI and then transfected into CHO cells to obtain a cell line expressing VH protein and LDP-VL protein, which was named CHO-Anti-MSLN-LDP cell line;

[0066] (3) Cultivating the CHO-Anti-MSLN-LDP cell line and purifying the antibody from the culture supernatant;

[0067] (4) The antibody obtained in step (3) is co-incubated with the chromophore of lidamycin to obtain a conjugate through the affinity interaction between LDP and the chromophore of lidamycin.

[0068] The present invention also protects the use of any of the above conjugates in the preparation of medicines.

[0069] The present invention also protects a drug, the active ingredient of which is the conjugate.

[0070] Any of the above drugs is a drug for inhibiting tumors.

[0071] Any of the above drugs can be drugs that inhibit tumor cells.

[0072] Any of the above tumors may be a tumor that expresses or highly expresses mesothelin.

[0073] Any of the above tumors may be ovarian cancer, pancreatic cancer or lung cancer, etc.

[0074] Any of the above tumor cells is a tumor cell that expresses or highly expresses mesothelin.

[0075] For example, the tumor cells can be human ovarian cancer cells, human pancreatic cancer cells, or human large cell lung cancer cells.

[0076] For example, the tumor cells may be SKOV3 cells, AsPC-1 cells, OVCAR-3 cells, SW1990 cells or H460-1 cells.

[0077] The present invention utilizes hybridoma fusion technology to immunize BALB / C mice with mesothelin antigens to prepare immune spleen cells, which are then fused with SP2 / 0 cells to establish a library of hybridoma cell lines secreting anti-mesothelin monoclonal antibodies. From this library, the hybridoma cell line 3B7, which stably and efficiently secretes antibodies, was screened. Furthermore, the present invention utilized the hybridoma cell line 3B7 to scale up monoclonal antibody production through ascites induction, producing a high-titer, high-affinity, and highly specific murine anti-mesothelin monoclonal antibody. The amino acid sequence of this monoclonal antibody was also determined. Furthermore, the present invention humanized this murine monoclonal antibody. Given that mesothelin is a highly researchable new anti-tumor target, the anti-mesothelin monoclonal antibody prepared by the present invention can serve as an ideal carrier for targeted anti-tumor drugs, laying a solid foundation for the research and development of targeted anti-tumor drugs and possessing promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The antibody concentrations are those prepared from the eight monoclonal hybridoma cell lines that can highly express anti-mesothelin antibodies in Example 1.

[0079] Figure 2 This is the result of detecting the antibody titer in Example 2.

[0080] Figure 3 The results of antibody purity detection by HPLC in Example 2 are shown.

[0081] Figure 4 This is the result of electrophoresis detection of antibody purity in Example 2.

[0082] Figure 5 This is the result of detecting the expression of mesothelin in the test cells in Example 3.

[0083] Figure 6 This is the result of flow cytometry detection of the ability of antibodies to bind to tumor cells in Example 3.

[0084] Figure 7 This is the result of confocal immunofluorescence detection of anti-mesothelin antibody in Example 3 (OVCAR-3 cells).

[0085] Figure 8 This is the result of confocal immunofluorescence detection using anti-mesothelin antibody in Example 3 (SKOV3 cells).

[0086] Figure 9 The results of in vivo imaging of mice in Example 3 to monitor the ability of antibodies to target tumors (OVCAR-3 cells).

[0087] Figure 10 The results of in vivo imaging of mice in Example 3 to monitor the ability of antibodies to target tumors (AsPC-1 cells).

[0088] Figure 11 The results of in vivo imaging of mice in Example 3 to monitor the ability of antibodies to target tumors (SKOV3 cells).

[0089] Figure 12 The results of the binding ability test of 3B monoclonal antibody to human tumor tissue chip in Example 3 are shown.

[0090] Figure 13 Schematic diagram of the structure of the recombinant plasmid pIZDHL-MSLN-IgG-LDP in Example 5.

[0091] Figure 14 Schematic diagram of the assembly of Anti-MSLN-LDP protein and lidamycin chromophore AE in Example 5.

[0092] Figure 15 This is the result of the killing activity of the antibody-lidamycin conjugate against tumor cells in Example 5. DETAILED DESCRIPTION

[0093] The present invention will be described in further detail below with reference to specific embodiments. The examples provided are intended solely to illustrate the present invention and are not intended to limit its scope. The following examples are intended to serve as a guide for further improvements by persons of ordinary skill in the art and do not in any way limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods and were performed according to techniques or conditions described in literature in the art or according to product specifications. Materials and reagents used in the following examples, unless otherwise specified, are commercially available. Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, the PBS buffer used in the examples is pH 7.4, 0.01 M PBS buffer. PBST solution: 500 μl of Tween 20 was added to 1000 ml of PBS buffer. Human mesothelin protein: Beijing Sino Biological Technology Co., Ltd., Cat. No. 13128-H08H1. SKOV3 cells: human ovarian cancer cells. AsPC-1 cells: human pancreatic cancer cells. OVCAR-3 cells: human ovarian cancer cells. SW1990 cells: human pancreatic cancer cells. H460 cells: human large cell lung cancer cells.

[0094] In the embodiment, the antibody titer and antibody concentration were detected by ELISA, and the specific method is as follows:

[0095] ① Dilute human mesothelin to 2 μg / mL with PBS buffer, then add to a 96-well plate (100 μL per well), incubate overnight at 4°C, and then wash three times with PBST solution (add 200 μL to each well each time, then shake for 3 minutes, and then discard the supernatant).

[0096] ②After completing step ①, add 2% BSA blocking solution (200 μL per well), incubate at 37°C for 2 hours, and then wash three times with PBST solution (add 200 μL per well each time, shake for 3 minutes, and discard the supernatant). 2% BSA blocking solution: Dissolve 2g BSA in 100ml PBS buffer.

[0097] ③After completing step ②, add the test sample (100 μL per well), incubate at 37°C for 2 h, and then wash three times with PBST solution (add 200 μL to each well each time, then shake for 3 minutes, and then discard the supernatant).

[0098] ④ After completing step ③, add secondary antibody working solution (100 μL per well), incubate at 37°C for 1 hour, and then wash three times with PBST solution (add 200 μL per well each time, shake for 3 minutes, and discard the supernatant). Secondary antibody working solution: Dilute horseradish peroxidase-conjugated goat anti-mouse IgG (Fc-specific) to 1000 volumes with PBS buffer. Horseradish peroxidase-conjugated goat anti-mouse IgG: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Cat. No. ZB-2305.

[0099] ⑤After completing step ④, add TMB colorimetric solution and incubate at room temperature for 30 minutes.

[0100] ⑥ After completing step ⑤, add 2M H2SO4 solution to terminate the reaction, and then use a microplate reader to detect the absorbance value at 450nm.

[0101] The test sample is the test antibody or the dilution of the test antibody (PBS buffer is used as the dilution solvent). Two replicate wells are set for the test sample.

[0102] Substitute the test sample with the standard solution and follow the above procedure to create a standard curve using absorbance and antibody concentration as variables. Set up three replicate wells for the standard solution. Standard solution: 200 μg / mL of SANTA mesothelin monoclonal antibody, first concentrated using an ultrafiltration tube, then diluted with PBS buffer to 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.13 μg / mL, 0.06 μg / mL, or 0.03 μg / mL. SANTA mesothelin monoclonal antibody (mesothelin mouse chimeric antibody): Santa Cruz Biotechnology, Cat. No. sc-271540.

[0103] According to the absorbance value, the titer of the test antibody can be obtained.

[0104] By substituting the absorbance value into the standard curve, the concentration of the test antibody can be calculated.

[0105] Example 1. Acquisition and preservation of hybridoma cells

[0106] 1. Obtaining hybridoma cells

[0107] 1. Animal immunization

[0108] Mesothelin antigen is human mesothelin protein. The mesothelin antigen solution is obtained by dissolving human mesothelin protein in PBS buffer.

[0109] At week 0, for the first immunization, equal volumes of mesothelin antigen solution and complete Freund's adjuvant were mixed and completely ultrasonically emulsified under ice bath conditions (3 min, ultrasound frequency less than 30%, 2 s intervals, 2 s pauses). Each mouse was intraperitoneally injected with 200 μL, equivalent to a total antigen amount of 50 μg / mouse.

[0110] Three weeks later, for the second immunization, the mesothelin antigen solution was mixed with equal volumes of incomplete Freund's adjuvant and completely emulsified by ultrasonication under ice bath conditions. Each mouse was intraperitoneally injected with 200 μL of the solution, equivalent to a total antigen volume of 25 μg per mouse.

[0111] 6 weeks later, the third immunization was performed with the same procedure as the second immunization.

[0112] At 7 weeks, the serum antibody titer of mice was tested by ELISA method, and the serum titer reached one million.

[0113] See Table 1 for exemplary results.

[0114] Table 1

[0115]

[0116] At 9 weeks, booster immunization was performed. Each mouse was intraperitoneally injected with 200 μl of mesothelin antigen solution (containing 50 μg of antigen), and cell fusion was performed three days later.

[0117] 2. Myeloma cell activation

[0118] SP2 / 0 cells were inoculated on the back of BALB / c mice and the tumor mass was grown to 500 mm. 3 The tumor pieces were peeled off from both sides and ground into a cell suspension. Cell count and viability were measured using a cell counter. Myeloma cells with a survival rate greater than 90% could be fused.

[0119] 3. Preparation of B lymphocytes

[0120] BALB / c mice with serum antibody titers reaching the fusion threshold were selected. Eyeballs were removed to collect blood, and serum was separated and stored at -20°C until needed. The mice were sacrificed by cervical dislocation and disinfected by soaking in 75% alcohol for 5 minutes. Using sterile scissors and forceps in a dissecting dish in a cleanroom, the skin on the left abdomen of the mouse was cut open. Replace the sterile scissors and forceps and open the abdominal cavity. The spleen, located on the posterior side of the left abdomen, is a 3-4 cm long, dark red, tongue-shaped tissue. Remove the spleen and place it in a dish containing 10 ml of serum-free culture medium. Gently wash and carefully remove the surrounding connective tissue (taking care to maintain the integrity of the outer mucosal layer of the spleen). Using a 5 mL syringe needle, poke two or three holes in the curved end of the spleen, aspirate serum-free culture medium, and inject the culture medium into the spleen through the pointed end of the spleen using a syringe. Each injection should last approximately 8 seconds to maintain a constant pressure and flow rate to fully flush out the B lymphocytes in the spleen. Rinse 3-5 times until the spleen turns white. Collect the B cell suspension and centrifuge at 1000 rpm for 5 minutes. Resuspend in serum-free medium and count the cells. Usually, 10 cells can be collected from one mouse. 8 B lymphocytes.

[0121] 4. Cell fusion

[0122] Take 1×10 8 splenocytes and 2×10 7 -5×10 7 Mix 1000 myeloma cells (SP2 / 0-Ag14) (usually in a ratio of 10:1-10:5) in a 50ml centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. Aspirate the supernatant and gently tap the bottom of the tube to loosen the pellet. Preheat the tube in a 40°C water bath until ready for use. Slowly and evenly add 1ml of 50% PEG-1450 solution (pH 8.0) preheated to 40°C over 45 seconds, gently swirling the tube to ensure that the PEG solution fully and evenly contacts the loosened cells. Pipette 1 ml of serum-free medium preheated to 40°C and slowly and evenly add it to the bottom of the centrifuge tube over 60 seconds, rotating the tube as you add. Pipette 5 ml of serum-free medium preheated to 40°C and slowly and evenly add it to the bottom of the centrifuge tube over 60 seconds, rotating the tube as you add. Pipette 10 ml of serum-free medium preheated to 40°C and slowly and evenly add it to the bottom of the centrifuge tube over 90 seconds, rotating the tube as you add. Repeat this process once. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Add 1640 complete medium supplemented with HAT medium, gently pipette to create a cell suspension, add the cells to a 96-well plate, and culture in a 5% CO2 incubator. After 5 days, replace half of the medium with HAT medium. After 7-10 days, replace the HAT medium with HT medium. After 14 days, replace the HAT medium with standard complete medium. Observe the growth of hybridoma cells. When they grow to more than 1 / 10 of the bottom area of the well, aspirate the supernatant for antibody detection, and perform a second antibody test at the same interval. Compare the two positive value results, pick out the wells with increased or unchanged positive values, preliminarily determine them as positive wells, and further screen the positive wells for cloning.

[0123] 5. Positive hybridoma screening and subcloning

[0124] Hybridoma cell rescreening and subcloning were performed using the dilution culture method. Mouse peritoneal cells were prepared. Hybridoma cell suspensions from positive wells were selected and diluted with HT medium containing 20% serum to different dilutions of 2.5, 15, and 50 cells per ml. The cells were plated onto 96-well plates at 0.2 ml per well, with the number of hybridoma cells per well being 0.5, 3, and 10, respectively. The cells were cultured at 37°C, 5% CO2 for 7-10 days until visible clones appeared. Observe under a microscope and select wells with only a single clone (a circular clone with a smooth arc of cell growth trajectory at the edge) for antibody detection. Cells from antibody-positive wells were subjected to the same dilution culture subcloning screening as above for 2-3 times, then expanded and frozen.

[0125] A total of 8 monoclonal hybridoma cell lines that can highly express anti-mesothelin antibodies were screened. The absorbance values of the corresponding antibodies at 450nm are shown in Table 2. The absorbance values were substituted into the standard curve to calculate the antibody concentration. The concentration of the corresponding antibodies is shown in Table 2. Figure 1 The cell line 3B7 had the highest antibody production capacity.

[0126] Table 2

[0127]

[0128] 6. Stability identification of hybridoma cell 3B7

[0129] Hybridoma cells 3B7 were cultured continuously, and the antibody titer in the cell culture supernatant was detected.

[0130] Table 3 shows the absorbance values at 450 nm for antibody titer analysis in cell culture supernatants during passaging. G1 represents passage 1, and so on. The results showed no significant changes in antibody titer during passaging or after resuscitation, demonstrating that hybridoma 3B7 cells can stably secrete anti-mesothelin monoclonal antibodies.

[0131] Table 3

[0132]

[0133]

[0134] 2. Deposit of Hybridoma Cells

[0135] Hybridoma cell 3B7, its full name is hybridoma cell QJW-520-3B7.

[0136] Hybridoma cell QJW-520-3B7 was deposited on June 25, 2019, at the China General Microbiology Center of Culture Collection (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) with the deposit registration number CGMCC No. 18168.

[0137] Example 2. Preparation of anti-mesothelin monoclonal antibodies

[0138] 1. Preparation of Monoclonal Antibodies

[0139] 1. 7-8 week old female BALB / c mice were intraperitoneally injected with Freund's incomplete adjuvant (500 μl / mouse) and housed normally for 7-10 days.

[0140] 2. After completing step 1, inject hybridoma cell suspension (200 μl / mouse) into the peritoneal cavity and feed normally for 7-10 days.

[0141] Hybridoma cell suspension: Resuspend hybridoma cells 3B7 in PBS buffer to a cell concentration of 1×10 7 pcs / ml.

[0142] 3. After completing step 2, collect ascites, centrifuge at 10,000 rpm for 30 minutes, and collect the supernatant.

[0143] 4. Take the supernatant obtained in step 3, filter it using a 0.45 μm filter membrane, and collect the filtrate.

[0144] 5. The filtrate obtained in step 4 was subjected to ammonium sulfate precipitation, and then purified using a Protein G (1 ml) affinity chromatography column to obtain a monoclonal antibody, designated as 3B7 monoclonal antibody.

[0145] 2. Preparation of control samples

[0146] SP2 / 0 cells were used instead of hybridoma cell line 3B7, and other procedures were the same as in step 1 to obtain a control sample.

[0147] 3. Detection of Antibody Titer

[0148] The titers of the 3B7 monoclonal antibody prepared in step 1 and the control sample prepared in step 2 were detected by ELISA.

[0149] See the results Figure 2 .

[0150] IV. Antibody Purity Identification

[0151] 1. HPLC method to detect antibody purity

[0152] Test antibody: 3B7 monoclonal antibody prepared in step 1.

[0153] Gel column: SEC S3000; flow rate: 1 ml / min; sample volume: 20 μl; detector: 280 nm;

[0154] Elution process: 0-16 min, the proportion of acetonitrile in the mobile phase increased from 5% to 95% in a gradient, with a total duration of 20 min;

[0155] The purity of the monoclonal antibody was calculated by the integrated peak area of the monoclonal antibody characteristic absorption at 280 nm.

[0156] See the results Figure 3 . The purity is above 95%.

[0157] 2. Electrophoresis to detect antibody purity

[0158] Test antibody: 3B7 monoclonal antibody prepared in step 1.

[0159] Take the test antibody and perform non-reducing protein electrophoresis and reducing protein electrophoresis respectively.

[0160] Electrophoresis gel: 12% separating gel, 5% stacking gel.

[0161] After Coomassie brilliant blue staining, the gel was photographed using a gel imaging system.

[0162] See the results Figure 4 .

[0163] 5. Antibody Class and Subclass Identification

[0164] Test antibody: 3B7 monoclonal antibody prepared in step 1.

[0165] The test antibody was taken and identified using the SBA Clonotyping System-HRP mouse monoclonal antibody typing kit.

[0166] The results are shown in Table 4. The heavy chain subtype of 3B7 monoclonal antibody is IgG1, and the light chain subtype is Kappa.

[0167] Table 4

[0168] Subtype <![CDATA[OD 450nm Value]]> P / N reaction IgA 0.1166 1.7613 － <![CDATA[IgG1]]> 1.7524 26.4713 + <![CDATA[IgG 2a ]]> 0.1004 1.5196 － <![CDATA[IgG 2b ]]> 0.1038 1.5680 － <![CDATA[IgG3]]> 0.1091 1.6480 － IgM 0.0757 1.1435 － Kappa 0.7811 11.7991 + Lambda 0.0965 1.4577 － control 0.0662 1.0000

[0169] VI. Amino Acid Sequence Determination of 3B7 mAb

[0170] RNA was extracted from hybridoma cell 3B7 and reverse transcribed to obtain a cDNA library. The nucleotide sequences encoding the antibody heavy chain and antibody light chain were obtained by PCR and sequencing, and then the amino acid sequences of the heavy chain and light chain were obtained.

[0171] The heavy chain variable region of the 3B7 monoclonal antibody is shown in Sequence 1 of the sequence listing (encoded by the DNA molecule shown in Sequence 2 of the sequence listing), and the light chain variable region of the 3B7 monoclonal antibody is shown in Sequence 3 of the sequence listing (encoded by the DNA molecule shown in Sequence 4 of the sequence listing).

[0172] Example 3. Performance of mouse monoclonal antibodies

[0173] 1. Detection of mesothelin expression in test cells

[0174] Test cells: SKOV3 cells, AsPC-1 cells, or OVCAR-3 cells.

[0175] The test cells were taken, total protein was extracted, and mesothelin was detected by western blot.

[0176] The primary antibodies used in western blot were the 3B7 monoclonal antibody prepared in step 1 of Example 2 or the mouse anti-β-actin antibody (Zhongshan Jinqiao, TA-09).

[0177] See the results Figure 5 .

[0178] 2. Flow cytometry to detect antibody binding ability to tumor cells

[0179] Test cells: SKOV3 cells, AsPC-1 cells, or OVCAR-3 cells.

[0180] 1. Take 5×10 6 100 μl of test cells were placed in an EP tube, washed with PBS buffer containing 2% FBS, and then resuspended with 100 μl of PBS buffer containing 2% FBS to obtain one cell suspension.

[0181] 2. Take the cell suspension and add the 3B7 monoclonal antibody prepared in step 1 of Example 2 to an antibody concentration of 10, 1, 0.1, 0.01, or 0.001 μg / ml. Take the cell suspension and add the control sample prepared in step 2 of Example 2 as a zero concentration control.

[0182] 3. After completing step 2, incubate at 4°C for 1.5 h, wash with PBS buffer containing 2% FBS, then add 200 μl FITC-labeled goat anti-mouse IgG secondary antibody (1:200 dilution), incubate at 4°C in the dark for 1 h, then wash three times with PBS buffer, and then detect by flow cytometry.

[0183] See the results Figure 6 3B7 mAb strongly bound to both OVCAR3 cells, which highly express mesothelin, and AsPC-1 cells, which moderately express mesothelin. The binding strength was dose-dependent with antibody concentration. 3B7 mAb bound only weakly to SKOV3 cells, which lowly express mesothelin. These results demonstrate that 3B7 mAb specifically binds to native mesothelin expressed on the surface of tumor cells.

[0184] 3. Confocal immunofluorescence detection of anti-mesothelin antibodies

[0185] Test cells: SKOV3 cells or OVCAR-3 cells.

[0186] This step is used to detect whether 3B7 mAb can bind to tumor cells and be transported to lysosomes through endocytosis.

[0187] 1. Prepare single cell suspension of test cells in logarithmic growth phase with a cell concentration of 1×10 4 pcs / 200μl.

[0188] 2. The single cell suspension was inoculated into an eight-well chamber (200 μl / well) and incubated at 37°C for 24 h. The 3B7 mAb (10 μg / mL, 200 μL / well) prepared in step 1 of Example 2 was then added and incubated at 4°C for 30 min or 37°C for 2 h. The 0.5 h incubation at 4°C was to detect mAb binding to the cell surface. The 2 h incubation at 37°C was to detect mAb endocytosis.

[0189] 3. Collect the cells, wash them three times with PBS buffer, then add 4% paraformaldehyde (200 μL / well) to fix for 15 min, then wash them three times with PBST solution, then add 0.2% Triton-X100 (200 μl Triton-X100 + 100 ml PBS buffer) 200 μL / well, permeabilize for 10 min, then wash them three times with PBST solution, then add 5% BSA solution, and block at 37°C for 30 min.

[0190] 4. Add rabbit anti-human lysosomal protein LAMP-1 antibody, incubate at 4°C overnight, wash 3 times with PBST solution, then add AF555 (red fluorescence) labeled donkey anti-rabbit secondary antibody (200 μL / well), incubate at 37°C for 2h, and add AF488 (green fluorescence) labeled goat anti-mouse secondary antibody (200 μL / well), incubate at 37°C for 30min, wash 5 times with PBST solution, add DAPI nuclear staining for 15min, wash 3 times with PBST solution, add anti-fluorescence quencher, and then observe under a confocal microscope.

[0191] 3B7 monoclonal antibody is labeled with green fluorescence. LAMP-1 antibody is labeled with red fluorescence. DAPI is used to stain nuclei and is labeled with blue fluorescence.

[0192] See the results Figure 7 and Figure 8 3B7 mAb binds to OVCAR-3 cells that overexpress mesothelin and is internalized and transported to lysosomes, emitting yellow fluorescence. No binding of 3B7 mAb to the cell membrane or internalization was observed in SKOV3 cells that underexpress mesothelin. These results suggest that 3B7 mAb can specifically target tumor cells that overexpress mesothelin and be transported to lysosomes via receptor-mediated endocytosis.

[0193] IV. In vivo imaging of mice to monitor the ability of antibodies to target tumors

[0194] Test cells: OVCAR-3 cells, AsPC-1 cells, or SKOV3 cells.

[0195] The 3B7 monoclonal antibody was labeled using the Dylight 680 antibody kit to obtain the labeled monoclonal antibody.

[0196] The test cells were inoculated into the axilla of BALB / c nude mice (6×10 4 cells / mouse), and the mice were raised until the tumor volume reached 200-300 cm 2 , then the labeled monoclonal antibody (20 mg / kg) was injected into the tail vein, and in vivo imaging was performed at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 24 h, 30 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, 120 h, 132 h, 144 h, 156 h, 168 h, 192 h, 216 h, 240 h, 264 h, 288 h, 312 h, 336 h, and 360 h respectively.

[0197] The results of OVCAR-3 cells are shown in Figure 9 The results of AsPC-1 cells are shown in Figure 10 The results of SKOV3 cells are shown in Figure 11 .

[0198] In the OVCAR-3 ovarian cancer xenograft model, DyLight 680-labeled 3B7 mAb began to accumulate in the tumor site 6 hours after administration, with peak fluorescence enrichment between 60 and 180 hours, and this fluorescence enrichment in the tumor site persisted for more than 15 days. In the AsPC-1 pancreatic cancer cell xenograft model, DyLight 680-labeled 3B7 mAb began to accumulate in the tumor site 3 hours after administration, with peak fluorescence enrichment between 60 and 156 hours, and this fluorescence enrichment in the tumor site persisted for more than 15 days. In the SKOV3 ovarian cancer cell xenograft model, due to the low expression of mesothelin antigen in SKOV3 cells, DyLight 680-labeled 3B7 mAb did not begin to accumulate in the tumor site until 36 hours after administration, and the fluorescence intensity was relatively weak.

[0199] On day 15 of drug administration, the BALB / c nude mouse model was killed, the tumor mass was removed, and the heart, liver, spleen, lungs, kidneys, pancreas, large intestine, small intestine, femur and stomach were taken and placed under a fluorescence imaging device for detection. The results showed that the isolated tumor still had a strong fluorescence signal, while other tissues and organs had no obvious fluorescence intensity, indicating that the 3B monoclonal antibody can be specifically and durably enriched in the tumor site.

[0200] 5. Determination of the binding ability of 3B monoclonal antibody to human tumor tissue microarray

[0201] Ovarian cancer tissue chip (product number: HOvaC151Su01) and pancreatic cancer tissue chip (product number: OD-CT-DgPan03-002) were purchased from Shanghai Xinchao Biotechnology Co., Ltd.

[0202] The main steps of immunohistochemical staining include: drying, dewaxing, antigen retrieval, endogenous peroxidase blocking, primary antibody incubation, secondary antibody incubation, DAB color development, hematoxylin counterstaining, and sealing.

[0203] See the results Figure 12 The binding activity of 3B7 monoclonal antibody to human ovarian and pancreatic cancer tumor tissues was significantly different from that to the corresponding adjacent tissues, with positive binding to tumor tissues and negative binding to adjacent tissues. This indicates that 3B7 monoclonal antibody can specifically bind to human ovarian and pancreatic cancer tissues.

[0204] Example 4. Preparation of humanized monoclonal antibodies

[0205] 1. Humanization of mouse monoclonal antibodies

[0206] Based on the sequence of 3B7 monoclonal antibody, humanized design was performed to obtain the humanized monoclonal antibody sequence.

[0207] The humanized monoclonal antibody was designated h3B7. The heavy chain of h3B7 is represented by Sequence 5 of the sequence listing (encoded by the DNA molecule represented by Sequence 6 of the sequence listing), and the light chain is represented by Sequence 7 of the sequence listing (encoded by the DNA molecule represented by Sequence 8 of the sequence listing).

[0208] 2. Preparation of humanized monoclonal antibodies

[0209] h3B7 monoclonal antibody was prepared (Beijing Anbiqi Biotechnology Co., Ltd.). The h3B7 monoclonal antibody has a heavy chain as shown in Sequence 5 of the sequence listing (encoded by the DNA molecule as shown in Sequence 6 of the sequence listing), and a light chain as shown in Sequence 7 of the sequence listing (encoded by the DNA molecule as shown in Sequence 8 of the sequence listing).

[0210] The method for preparing h3B7 monoclonal antibody is briefly described as follows: an exogenous DNA molecule is inserted into the pcDNA3.1-IgG1Fc expression vector to obtain a heavy chain expression vector (which contains the DNA molecule shown in Sequence 6 of the sequence listing and expresses the heavy chain shown in Sequence 5 of the sequence listing); an exogenous DNA molecule is inserted into the pcDNA3.1-IgKc expression vector to obtain a light chain expression vector (which contains the DNA molecule shown in Sequence 8 of the sequence listing and expresses the light chain shown in Sequence 7 of the sequence listing); the heavy chain expression vector and the light chain expression vector are co-transfected into 293F-SVP16 cells, and the expression vector is expressed using FreeStyle TMCulture the cells in 293 expression medium (Thermo Fisher Scientific (China) Co., Ltd., Cat# 12338018). The supernatant was collected and filtered through a 0.45 μm filter. The filtrate was purified using Protein A magnetic beads to obtain a monoclonal antibody solution, i.e., the h3B7 monoclonal antibody solution. The pcDNA3.1-IgG1Fc expression vector, pcDNA3.1-IgKc expression vector, and 293F-SVP16 cells were all products of Ambigene.

[0211] 3. Biacore method for detecting antibody-antigen affinity

[0212] Test antibody: 3B7 monoclonal antibody prepared in step 2 of Example 2 or h3B7 monoclonal antibody prepared in step 2 of this example.

[0213] BiaCoreT200 was used to detect the affinity of the test antibody to the target protein (i.e., human mesothelin protein).

[0214] 3B7 monoclonal antibody concentration: 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 μg / ml.

[0215] h3B7 monoclonal antibody concentration: 10 / 5 / 2.5 / 1.25 / 0.625 / 0.3125 μg / ml.

[0216] Flow rate: 30 μl / min.

[0217] Combination time: 300s.

[0218] Dissociation time: 300s.

[0219] Regeneration reagent: Glycine 2.0.

[0220] The results are shown in Table 5.

[0221] Table 5

[0222] 3B7 monoclonal antibody h3B7 monoclonal antibody <![CDATA[Ka(M -1 s -1 )]]> 2746 <![CDATA[1.231×10 5 ]]> <![CDATA[kd(s -1 )]]> <![CDATA[4.181×10 -5 ]]> <![CDATA[3.394×10 -4 ]]> KD(M) <![CDATA[1.552×10 -8 ]]> <![CDATA[2.757×10 -9 ]]>

[0223] Example 5. Construction, expression and purification of fusion protein anti-MSLN-LDP

[0224] 1. Construction of recombinant plasmid

[0225] Plasmid pIZDHL is described in the following literature: Xiao-Yun Liu, et al. Chimetric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antimemoral activity against human B-cell lymphoma and pre-B acute lymphoblastic leukemia cell lines. Int J Cancer, 2011, 129(2): 497-506. Plasmid pIZDHL contains genes encoding the antibody heavy chain constant region and the antibody light chain constant region.

[0226] The small fragment between the NheI and XhoI restriction enzyme recognition sequences of the plasmid pIZDHL was replaced with the DNA molecule shown in sequence 10 of the sequence listing, and the small fragment between the MluI and BsiWI restriction enzyme recognition sequences was replaced with the DNA molecule shown in sequence 12 of the sequence listing, while keeping the other sequences unchanged, to obtain the recombinant plasmid pIZDHL-MSLN-IgG-LDP. Figure 13 .

[0227] The DNA molecule shown in Sequence No. 10 in the Sequence Listing encodes the protein shown in Sequence No. 9 in the Sequence Listing. The protein shown in Sequence No. 9 in the Sequence Listing is a VH protein (heavy chain variable region of a humanized full-length heavy chain).

[0228] The DNA molecule shown in Sequence 12 of the sequence listing encodes the protein shown in Sequence 11 of the sequence listing. In the protein shown in Sequence 11 of the sequence listing (LDP-VL protein), amino acid residues 1-21 constitute the signal peptide, amino acid residues 22-131 constitute the lidamycin cofactor protein LDP, and amino acid residues 140-243 constitute the VL protein (the light chain variable region of the humanized full-length light chain).

[0229] The recombinant plasmid pIZDHL-MSLN-IgG-LDP expressed the heavy chain (having VH protein and heavy chain constant region) and the LDP-fused light chain (having LDP, VL protein and light chain constant region), which self-assembled into an antibody protein named Anti-MSLN-LDP protein.

[0230] 2. Expression of the fusion protein Anti-MSLN-LDP

[0231] 1. The recombinant plasmid pIZDHL-MSLN-IgG-LDP was digested with pvuI to obtain the linearized plasmid.

[0232] 2. Transfect CHO cells (plated into 6-well plates the day before) with the linearized plasmid obtained in step 1, culture for 48 h, then collect the cells and digest them with trypsin.

[0233] 3. The digested cells obtained in step 2 were inoculated into 96-well plates (approximately 10 3 cells / well), cultured for 24 h, and then screened with IMDM medium containing 2 μg / ml bleomycin and 10% dialyzed fetal bovine serum to obtain a cell line expressing Anti-MSLN-LDP protein, named CHO-Anti-MSLN-LDP cell line.

[0234] 3. Purification of the fusion protein Anti-MSLN-LDP

[0235] CHO-Anti-MSLN-LDP cell line was inoculated into T75 cell culture flasks and cultured in IMDM medium containing 10% dialyzed fetal bovine serum until the cell density reached above 90%, and then replaced with serum-free medium (CD Opti CHO TM Medium, Gibco) was continuously cultured for 10 days, and then the liquid phase in the culture flask was transferred to a 50 ml centrifuge tube, centrifuged at 4°C and 8000 rpm for 15 min, the supernatant was collected, filtered through a 0.45 μm membrane and the filtrate was collected, then centrifuged and concentrated using a 30,000 MW ultrafiltration tube, and then purified using a Protein G affinity chromatography column, and then desalted using a desalting column, and then centrifuged and concentrated again using a 30,000 MW ultrafiltration tube to obtain a protein solution, which was named Anti-MSLN-LDP protein solution.

[0236] 4. Preparation of Antibody-Lidamycin Conjugates

[0237] 1. Take Lidamycin and separate the active chromophore AE using a C4 column (the mobile phase is water: acetonitrile: trifluoroacetic acid = 77.9%: 22%: 0.1%, volume ratio, and the absorbance at 350 nm is detected), and collect the chromophore AE.

[0238] 2. Mix the Anti-MSLN-LDP protein prepared in step 3 with the chromophore AE at a molecular ratio of 1:3, place on a shaker and shake slowly, and react at 4°C in the dark for 12-16 hours.

[0239] 3. After completing step 2, ultrafiltration centrifuge the mixture of the two at 4°C and 3500rpm for 4-6 times (stop ultrafiltration when the absorption value of the chromophore is not detected in the ultrafiltered solution), and collect the filtrate. The filtrate contains the antibody-lidamycin conjugate (i.e., the Anti-MSLN-LDP protein forms a conjugate with the chromophore AE of lidamycin by virtue of the binding effect of the cofactor protein LDP of lidamycin and the chromophore AE of lidamycin), so the filtrate is also named as the antibody-lidamycin conjugate solution. The assembly diagram of the Anti-MSLN-LDP protein and the lidamycin chromophore AE is shown in Figure 14 .

[0240] 5. Cytotoxicity of Antibody-Lidamycin Conjugates against Tumor Cells

[0241] Test cells: OVCAR-3 cells, AsPC-1 cells, SW1990 cells, and H460 cells.

[0242] Take the test cells in the logarithmic growth phase, count them and inoculate them into a 96-well plate (3000 / well), add the antibody-lidamycin conjugate (set different conjugate concentrations) or lidamycin (set different lidamycin concentrations) prepared in step 4 the next day, culture for 48 hours, add CCK-8 reagent, measure its absorbance at 450nm on an enzyme reader, and detect the killing activity of the antibody-lidamycin conjugate or lidamycin on tumor cells.

[0243] See the results Figure 15 At consistent concentrations (equal molar masses), the antibody-lidamycin conjugate exhibited a similar inhibition rate on tumor cell proliferation as lidamycin, and both exhibited similar cytotoxic effects on MSLN-positive tumor cells, indicating that the ADC constructed with the flexible linker peptide did not affect the assembly of the lidamycin cofactor protein LDP and AE, nor did it affect the activity of AE. Therefore, the antibody-lidamycin conjugate not only possesses the affinity and targeting properties of Anti-MSLN-IgG for the MSLN antigen, but also possesses the highly effective cytotoxic activity of LDM against tumor cells.

[0244] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. An anti-human mesothelin monoclonal antibody or an antigen-binding portion thereof, characterized in that: The monoclonal antibody or antigen-binding portion thereof contains a H The heavy chain variable region and the name V L The light chain variable region, the V H and V L are composed of a complementary region and a framework region; the V H and the V L The complementary regions of the determinants are composed of CDR1, CDR2 and CDR3; The V H The amino acid sequence of CDR1 is shown in positions 31-35 of SEQ ID No: 5; The V H The amino acid sequence of CDR2 is shown in positions 50-66 of SEQ ID No: 5; The V H The amino acid sequence of CDR3 is shown in SEQ ID No: 5, positions 99-109; The V L The amino acid sequence of CDR1 is shown in positions 24-34 of SEQ ID No: 7; The V L The amino acid sequence of CDR2 is shown in positions 50-56 of SEQ ID No: 7; The V L The amino acid sequence of CDR3 is shown in positions 89-97 of SEQ ID No:

7.

2. The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein: The heavy chain variable region thereof is shown as positions 1-120 of SEQ ID No: 5, and the light chain variable region thereof is shown as positions 1-104 of SEQ ID No:

7.

3. The monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein: The heavy chain variable region thereof is shown as positions 20 to 139 of SEQ ID No: 1, and the light chain variable region thereof is shown as positions 21 to 127 of SEQ ID No:

3.

4. Hybridoma cells or monoclonal antibodies secreted by the hybridoma cells; The hybridoma cell is hybridoma cell QJW-520-3B7, and its deposit registration number is CGMCC No.18168.

5. Biological materials, which are (a), (b), (c), (d), or (e) below: (a) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3; (b) an expression cassette comprising the nucleic acid molecule of (a); (c) a recombinant vector having the nucleic acid molecule described in (a); (d) a recombinant microorganism having the nucleic acid molecule described in (a); (e) A transgenic cell line having the nucleic acid molecule described in (a).

6. Use of the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3, or the hybridoma cell according to claim 4, or the monoclonal antibody according to claim 4, or the biomaterial according to claim 5, which is as follows (I) or (II) or (III) or (IV): (I) Use in the preparation of vectors for targeting tumors or tumor cells; (II) Application in the preparation of products for detecting mesothelin; (III) Use in the preparation of products for detecting tumor tissues or tumor cells; (IV) Use in the preparation of products for inhibiting tumors or inhibiting tumor cell proliferation; The tumor is ovarian cancer, pancreatic cancer or lung cancer.

7. A product, the active ingredient of which comprises the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3 or the monoclonal antibody according to claim 4; The product is as follows (i) or (ii) or (iii): (i) vectors for targeting tumors or tumor cells; (ii) Products for the detection of mesothelin; (iii) Products used for detecting tumor tissues or tumor cells.

8. A conjugate of an antibody and a drug; the antibody is the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 3 or the monoclonal antibody according to claim 4; and the drug is lidamycin.

9. Use of the conjugate according to claim 8 in the preparation of a drug; the drug is a drug for inhibiting tumors or inhibiting tumor cell proliferation; the tumor is ovarian cancer, pancreatic cancer or lung cancer.

10. A medicine, the active ingredient of which comprises the conjugate according to claim 8.

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